Symmetry of Charge Partitioning in Collisional and UV Photon-Induced Dissociation of Protein Assemblies

Abstract

Tandem mass spectrometry can provide structural information on intact protein assemblies, generating mass fingerprints indicative of the stoichiometry and quaternary arrangement of the subunits. However, in such experiments, collision-induced dissociation yields restricted information due to simultaneous subunit unfolding, charge rearrangement, and subsequent ejection of a highly charged unfolded single subunit. Alternative fragmentation strategies can potentially overcome this and supply a deeper level of structural detail. Here, we implemented ultraviolet photodissociation (UVPD) on an Orbitrap mass spectrometer optimized for native MS and benchmark its performance to HCD fragmentation using various protein oligomers. We investigated dimeric β-lactoglobulin, dimeric superoxide dismutase, dimeric and tetrameric concanavalin A, and heptameric GroES and Gp31; ranging in molecular weight from 32 to 102 kDa. We find that, for the investigated systems, UVPD produces more symmetric charge partitioning than HCD. While HCD spectra show sporadic fragmentation over the full protein backbone sequence of the subunits with a bias toward fragmenting labile bonds, UVPD spectra provided higher sequence coverage. Taken together, we conclude that UVPD is a strong addition to the toolbox of fragmentation methods for top-down proteomics experiments, especially for native protein assemblies.

Self-assembly of toroidal proteins explored using native mass spectrometry

“…That is, the energy of unfolding subunits is greater than the energy required to disrupt the dimer–dimer interface of the dodecamers. This symmetric dissociation pathway has been observed in experiments employing collision-induced dissociation, 34 surface-induced dissociation, 35 , 36 and ultra-violet photodissociation. 37 Dissociation via these symmetric pathways is usually related to structural features of the protein, such as differing protein–protein interface sizes.…”

Ion traps in modern mass spectrometry

“…Also, the technology could be used in combination with light spectroscopy of cold ions for the analysis of molecular structures, allowing for example separation and quantitation of isomers . These experiments make a significant step in fidelity of structure determination comparing, for example, even to such promising fragmentation methods as ultraviolet photodissociation already combined with Orbitrap MS . It should be noted that in such combinations the Orbitrap analyzer remains just a detector while manipulations with ions are performed in external linear RF traps as described in section 2.…”

Imaging of protein distribution in tissues using mass spectrometry: An interdisciplinary challenge

“…To circumvent this issue, a "pseudo-MS 3 " approach, also known as "T 3 -sequencing", has been developed to improve MALDI-ISD in proteins 168,169 . In this approach, the fragments produced by Another approach to identify proteins uses fragmentation methods in mass spectrometers applied in "top-down" protein analysis such as ETD, ECD, or UVPD [172][173][174][175] . These might be applicable to topdown identification approaches in MSI, although the speed and sensitivity are currently not yet compatible with MSI.…”